Viruses in the Reoviridae family including reoviruses and orbiviruses when passaged serially in cell cultures at a high moi have been demonstrated to generate defective interfering (DI) particles. Rotaviruses are distinct in this aspect from other members of the Reoviridae family. Rotaviruses when passaged serially in cell cultures at a high moi generate viruses bearing rearranged gene(s) stemming from intragenic recombination. The detection of rotavirus DI particles has not been reported. Interestingly, just like DI particles of reoviruses, rotaviruses bearing an intragenic recombinant gene(s) interfere with the replication of wild-type viruses, however, unlike reovirus DI particles these rotaviruses bearing rearranged intragenic recombinant genes are viable. In order to (i) study molecular and biologic characteristics of a virus bearing rearranged gene(s); and (ii) pursue a possible use of such viruses as vaccine candidates, we passaged serially a porcine rotavirus (SB1A strain) in MA104 cells at a high moi and detected three progeny viruses each of which bore distinct rearranged gene(s). Northern blot analysis of passage 67 lysates demonstrated the presence of two different rearranged NSP3 genes and one rearranged gene that possessed both NSP2 and NSP5 specificities. Sequencing revealed that (i) the rearranged NSP3 genes consisted of a partial duplication in a head-to-tail orientation without altering the NSP3 ORF (an intragenic recombination) and (ii) the recombinant gene, 1647 nucleotides (nt) in length, contained the truncated NSP2 gene inserted into the NSP5 gene at nt 332. The expression of the NSP5 and fusion proteins by the recombinant gene was undetectable by Western blotting. Viruses bearing the rearranged NSP3 gene were viable, however, viruses bearing the recombinant NSP2-NSP5 gene appeared to be nonviable and to interfere with the replication of viable viruses. This is the first report describing an intergenic recombination of rotavirus genes.[unreadable] We also passaged serially a murine rotavirus (EB strain) in primary African green monkey kidney cells or in an established cell line MA104 at a high moi and obtained (i) two progeny viruses each bearing a distinct rearranged gene 5 encoding NSP1 protein; and (ii) three mutant viruses each bearing a distinct rearranged gene 7 encoding NSP3 protein.